Various publications, including patents, published applications, technical articles and scholarly articles are cited throughout the specification. Each of these cited publications is incorporated by reference herein, in its entirety and for all purposes.
Sodium plays an important role in the body's metabolism, including, among other things, electrical impulse transmission and fluid and electrolyte homeostasis. In addition, sodium contributes to the development and stability of flavor in the various foods ingested by animals, particularly by humans. The sodium ion can inhibit the bitter taste of some stimuli, thereby modifying the taste of food. This inhibitory effect of sodium on bitter taste does not depend on the saltiness of the compound containing the sodium ion, but rather depends on the concentration of the sodium ion.
Excess intake of sodium, however, has been implicated in various disease states, including gastric cancer and hypertension. Hypertension is a major risk factor for heart disease, stroke, and kidney disease. Because of the potential negative health effects of excess sodium consumption, the United States FDA recommends that adults limit their intake to less than 2400 milligrams of sodium per day. Nevertheless, Americans generally far exceed this recommended allowance. As such, various medical and scientific groups have recommended drastic reductions in sodium intake.
To further the goal of reduced sodium intake, numerous salty taste mimics and salty taste enhancers have been developed. In general, such mimics have not proven commercially viable as they lack the clean saltiness of sodium chloride, and most do not affect food flavor as sodium salt does.
The dearth of mimics of salty taste, commonly known as salt substitutes, reflects the extreme structural specificity of the taste receptor. As far is known, only sodium chloride (NaCl) and lithium chloride (LiCl) impart a true salty taste. Both heavier anions paired with Na and Li, and heavier cations paired with Cl tend to be bitter. The cation specificity suggests an ion channel, while the chloride effects suggests paracellular shunts. In addition, the concentration at which NaCl imparts a salty taste is above 50 mM, a concentration on the higher end of receptor processes. These two observations—the specificity for Na and Li, and the effective concentration range—are believed to be the key to discovering the mechanism of salty taste in humans.
Over the past two decades, numerous studies, both qualitative and quantitative, of salt-induced changes in neural activity in the presence or absence of specific inhibitors and enhancers have led to the supposition that an epithelial sodium channel (ENaC) acts as the primary receptor for saltiness (Brand et al. (1985) Brain Res. 334:207-14; Feigin et al. (1994) Am. J. Physiol. 266 (Cell Physiol):C1165-72; and, Brelin et al. (2006) Adv. Otorhinolaryngol. 63:152-90). While the ENaC serves as the salt receptor for many experimental animals (Halpern, B P (1998) Neurosci. Biobehav. Rev. 23(1):5-47), no conclusive evidence has emerged that the same holds true for human beings. Notably, the inability of amiloride to inhibit sodium-induced salty taste response in humans suggests that ENaCs are not involved in human salty taste recognition, at least to the extent observed in other animals.
Because of this discrepancy between human and animal models, the transduction mechanisms underlying the perception of salty taste in humans remain under investigation. Sufficient activation of the nerve eventually evokes the sensation of saltiness in the higher cortical areas (Schoenfeld, M A et al. (2004) Neuroscience. 127:347-53).
Because of the robust response shown to amiloride by taste cells of many rodents, the ENaCs in these cells are assumed to be located primarily at the apical membrane, above the level of the tight junctions. This location makes them susceptible to the action of drugs such as amiloride. It is assumed that amiloride cannot pass the tight junctions. Augmenting the direct mechanism at the apical membrane is a paracellular shunt pathway into the basolateral area of taste buds below the tight junction level (Mierson, S et al. (1996) J. Neurophysiol. 76:1297-309). Since sodium can pass the tight junctions, the paracellular mechanism should result in an amiloride insensitive salty taste response. The human salty response may be amiloride-insensitive because the vast majority of taste cell ENaCs are located below these tight junctions. Other mechanisms for salt perception may exist. These could be entirely different from the ENaC, or an alternative manifestation of the ENaC due to sodium load or hormonal influences on ENaC expression or composition.
ENaCs comprise a family of cation channel proteins mediating sodium permeation in epithelia (Mano, I et al. (1999) Bioessays 21:568-78). Expression cloning originally demonstrated that there are three homologous genes, each encoding one of the three subunits of the channel—i.e., alpha (α), beta (β) and gamma (γ) (Canessa, C M et al. (1994) Nature 367:463-7). Co-expression of all three subunits is essential for maximal Na+ channel activity, although the alpha subunit by itself produces a small current. A fourth subunit, delta (δ) was later cloned and shown to be similar to the alpha subunit both structurally and functionally, albeit with a 30-fold lower affinity for amiloride (Waldmann et al. (1995) J. Biol. Chem. 270:27411-4). This lower amiloride sensitivity is assumed to be reflected in a motif called the PreMR2 sequence. The transmembrane topology of the ENaCs comprises two hydrophobic transmembrane domains flanking a long extracellular loop, with intracellular amino and carboxyl termini. The subunit stoichiometry of the ENaCs may be species-specific and tissue-specific, since there is evidence for an α2βγ configuration in rats (Firsov et al. (1998) EMBO J. 17:344-52) and an (α)1β(1)γ(1) arrangement in humans (Staruschenko, A (2005) Biophys. J. 88:3966-75).
For improved health and wellness, there is a need to diminish sodium intake. This need must be balanced with the desire for the taste of sodium, and the ability of sodium to impart improved flavor in food. One attractive means to diminish dietary sodium without sacrificing sodium flavor is to use modulators of salty taste. Thus, there is a need to establish the definitive receptor for salty taste perception and for a means to identify modulators of salty taste perception.